Project Summary/Abstract Nonadditive interactions between mutant sites in the same protein can dictate the selective accessibility of alternative mutational pathways through sequence space, and therefore represent a potential source of contingency in adaptive protein evolution. If the fitness effects of mutations are dependent on genetic background, the accumulated history of substitutions in the past may influence the set of allowable mutations in the future, and particular adaptive outcomes may therefore be contingent on ancestral starting points. For this reason, a particular adaptive solution may be more accessible to species A than to species B (and vice versa) simply due to evolved differences in genetic background. The goal of this project is to experimentally test the role of such contingency in the adaptive evolution of the transmembrane Na+,K+-ATPase protein in colubroid snakes. Specifically, I will combine site-directed mutagenesis experiments with ancestral protein resurrection to examine the molecular basis of target-site insensitivity of Na+,K+-ATPase, a change in protein function that mediates resistance to cardiac glycosides. In several different families of snakes, resistance to cardiac glycosides has evolved independently in species that prey on toxic toads. Among different species of toad-eating snakes, and among numerous other animal taxa that have evolved resistance to cardiac glycosides, target-site insensitivity of Na+,K+-ATPase is attributable to various combinations of divergent, convergent, and parallel amino acid substitutions, providing a rich body of comparative data. Using ancestral protein resurrection in conjunction with a protein engineering approach based on site-directed mutagenesis, I will compare the functional effects of causative substitutions on clade-specific ancestral backgrounds and on the background of a more ancient ancestor, shared by all clades. The specific aims of the project are to (1) identify the causative substitutions that confer resistance to cardiac glycosides in colubroid snakes and (2) to determine the extent to which the function-altering effects of these causative substitutions are dependent on the genetic context in which they occur. Together, accomplishing Aims 1 and 2 will reveal the molecular basis of a key physiological innovation and will provide general insights into the pathways by which such innovations evolve. The proposed research involves training with Dr. Jay Storz at University of Nebraska-Lincoln (UNL). Dr. Storz, a leader in the field of protein evolution, is well-known for taking a multi-disciplinary approach to gain a more complete understanding of the mechanisms governing evolution. The proposed training plan for this research would add a strong evolutionary biology component to my research repertoire, and as a result significantly increase the breadth of questions I will be able to address and collaborations I will be able to form in future work. Furthermore, UNL represents an ideal environment for postdoctoral training because it provides numerous key resources for career development and a highly productive and supportive environment.